As early as 1966, Skeggs described air-segmented continuous flow analysis. This approach to serial assays involved the introduction of successive samples into a length of tubing. Reagents are added at strategic points and mixing and incubation take place while the sample is on its way to the flow cell of the detector. Intermixing of adjacent samples is prevented by the introduction of air bubbles into the flow between samples. The first system was developed for the determination of glucose and urea in blood.
Flow injection analysis (FIA) represented a significant advancement in the automation of wet chemical analysis procedures. FIA has been described as a simple and versatile analytical technology for automating wet chemical analysis, based on the physical and chemical manipulation of a dispersed sample zone formed from the injection of the sample into a flowing carrier stream and detection downstream. FIA avoids the use of bubbles to separate samples. Examples of FIA systems and apparatus are described in U.S. Pat. Nos. 4,952,372 and 5,695,720, which are incorporated herein in their entirety by reference thereto.
The power of FIA as an analytical tool lies in its ability to combine sampling, sample processing, and detection in a wide variety of different ways to create a broad range of different methodologies, and perform these methodologies rapidly and automatically with minute amounts of sample. The device most commonly used to measure out the sample and insert it into the FIA carrier stream is a two-position sample injection valve. Peristaltic pumps are frequently used to propel streams in the flow injection manifold. Many detection techniques have been applied to FIA.
Sequential Injection Analysis (SIA) was developed in response to the requirement for a more rugged and simple apparatus suitable for automation within industrial applications. Like FIA, SIA is an automated approach to sample handling that allows the convenient automation of different manual wet-chemistry procedures to provide rapid, precise, and accurate measurements. Small solution zones are manipulated under controlled dispersion conditions in narrow bore conduits, typically tubing. SIA systems and apparatus are discussed in U.S. Pat. No. 5,849,592, which is incorporated herein in its entirety by reference thereto.
While, like FIA, SIA is fundamentally dependent on the dispersion of zones in a flowing stream, conceptually, the practice of SIA is different from FIA. Where FIA is dependent on the physical dimensions of a sample loop, SIA makes use of an accurate metering pump to assemble a stack of precisely measured sample and reagent zones selected using a multi-position selection valve and a holding coil. Flow reversal and passage through the micro-bore tubing of the SIA manifold ensures intimate mixing of sample and reagent zones to form a detectable species. Different mixing strategies can be achieved using the same SIA system. Accordingly, SIA systems can easily and quickly switch between different tests of sample and reagent zones for a selected species. In FIA, swapping to a different test frequently requires a time and labor intensive change to the manifold hardware. In SIA on the other hand, this can be accomplished simply by a change in the flow-control sequence and in some cases the detector.
SIA has several advantages over FIA. Reagent use is drastically reduced. Typical FIA experiments make use of at least 1 ml of reagent per measurement. SIA typically makes use of less than 100 μl. Tubing manifolds are simple and robust typically comprising a pump, selection valve, and connected by a flow conduit such as micro-bore tubing. The same manifold can be used for widely different chemistries simply by changing the flow sequence rather than the plumbing. Analyzer maintenance is therefore simplified and the same manifold can be used for tests that require quite different sample handling strategies. The selection valve replaces the injection valve and provides a means for selecting different sample streams and calibrants. This enables a convenient means of swapping from one test to the next and automating the calibration of the apparatus.
A basic SIA system comprises a high precision pump to meter and propel sample and reagent zones within the system, a multi-position selection valve, micro bore tubing in the form of at least a holding coil and possibly a reaction coil or other suitable flow conduit, and a detector or detectors equipped with suitable flow through cells. Most often, a LED-based photometer is used as the detector, but the practice of SIA is not limited to these simple devices and transducers that make both physical and chemical measurements have been used. The selection valve has multiple ports that can be used for connection to selected reagents reservoirs, sample streams, calibration standards, and one or more detectors. While the flat plate rotary design of multi-position selection valves has been widely used, other stream selection manifolds could serve equally well. Because of their excellent accuracy and pulseless flow, syringe pumps are most frequently used in SIA.
Although the reagent can be included in the carrier stream, more often than not, the reagent is loaded as a separate zone. Then the syringe is filled with a simple carrier solution or chemical buffer solution. This carrier solution has several functions. It is used to propel reactants and reaction products in the flow manifold. The carrier solution is also used to create a chemical environment conducive to the measurement chemistry, e.g. by adjusting the pH or ionic strength of the reaction media. It also is useful in flushing the flow manifold so that there is no carry over or cross-contamination from one sample to the next. Because the detector is exposed to this clean solution for the majority of the time, fouling is minimized.
After the sample zone has been drawn up into the holding coil, the selection valve is advanced to a port connected to a reagent reservoir and a small reagent zone is drawn up into the holding coil. In this way it is possible to construct a stack of well defined zones which, when the sample and reagent are mixed together, give rise to a detectable species. Accurate measurement of sample and reagent zones necessitates repeatable and accurate control of the timing of component events. Once developed a suitable controller slavishly repeats the measurement sequence to give reproducible results. Different results can be achieved by changing the sequence of reagent and sample selection.
The successful implementation of SIA hinges on the reproducible mixing of a well-defined stack of reagent and sample zones. The mixing of these initially distinct zones is achieved through controlled dispersion in the reaction coil and reversal of the flow in the holding coil. Optimum sensitivity is achieved when mixing is maximized and axial dispersion is minimized. For this reason, reactors incorporated in the apparatus frequently make use of a geometric arrangement that ensures rapid and frequent changes in direction.
The existing art in the field of medical diagnostic tests is extensive. In this regard, enzyme-linked immunosorbent assays (ELISA) enjoy ubiquitous application. Countless methods and chemistries have been developed for a wide range of medical diagnostic tests and are available in kit form. Many of these rely on manual introduction of reagent components using hand held micropipettes. Robotic methods have been applied to the automation of these tests, and segmented-flow analysis techniques have been applied to medical diagnostic tests. While these methods and chemistries are highly beneficial for selected tests and techniques, advances in the automation and methods of medical diagnostic tests are still in need.